A massively multi-scale approach to characterizing tissue architecture by synchrotron micro-CT applied to the human placenta.

Multi-scale structural assessment of biological soft tissue is challenging but essential to gain insight into structure-function relationships of tissue/organ. Using the human placenta as an example, this study brings together sophisticated sample preparation protocols, advanced imaging and robust, validated machine-learning segmentation techniques to provide the first massively multi-scale and multi-domain information that enables detailed morphological and functional analyses of both maternal and fetal placental domains. Finally, we quantify the scale-dependent error in morphological metrics of heterogeneous placental tissue, estimating the minimal tissue scale needed in extracting meaningful biological data. The developed protocol is beneficial for high-throughput investigation of structure-function relationships in both normal and diseased placentas, allowing us to optimize therapeutic approaches for pathological pregnancies. In addition, the methodology presented is applicable in the characterization of tissue architecture and physiological behaviours of other complex organs with similarity to the placenta, where an exchange barrier possesses circulating vascular and avascular fluid spaces.

Using Scipion for stream image processing at Cryo-EM facilities.

Three dimensional electron microscopy is becoming a very data-intensive field in which vast amounts of experimental images are acquired at high speed. To manage such large-scale projects, we had previously developed a modular workflow system called Scipion (de la Rosa-Trevín et al., 2016). We present here a major extension of Scipion that allows processing of EM images while the data is being acquired. This approach helps to detect problems at early stages, saves computing time and provides users with a detailed evaluation of the data quality before the acquisition is finished. At present, Scipion has been deployed and is in production mode in seven Cryo-EM facilities throughout the world.

LepNet: The Lepidoptera of North America Network.

The Lepidoptera of North America Network, or LepNet, is a digitization effort recently launched to mobilize biodiversity data from 3 million specimens of butterflies and moths in United States natural history collections (http://www.lep-net.org/). LepNet was initially conceived as a North American effort but the project seeks collaborations with museums and other organizations worldwide. The overall goal is to transform Lepidoptera specimen data into readily available digital formats to foster global research in taxonomy, ecology and evolutionary biology.

A facility for the analysis of the electronic structures of solids and their surfaces by synchrotron radiation photoelectron spectroscopy.

A synchrotron radiation beamline in the photon energy range of 18-240 eV and an electron spectroscopy end station have been constructed at the 3 GeV Diamond Light Source storage ring. The instrument features a variable polarisation undulator, a high resolution monochromator, a re-focussing system to form a beam spot of 50 × 50 µm2, and an end station for angle-resolved photoelectron spectroscopy (ARPES) including a 6-degrees-of-freedom cryogenic sample manipulator. The beamline design and its performance allow for a highly productive and precise use of the ARPES technique at an energy resolution of 10-15 meV for fast k-space mapping studies with a photon flux up to 2 ⋅ 1013 ph/s and well below 3 meV for high resolution spectra.

Chemical imaging of single catalyst particles with scanning µ-XANES-CT and µ-XRF-CT.

The physicochemical state of a catalyst is a key factor in determining both activity and selectivity; however these materials are often not structurally or compositionally homogeneous. Here we report on the 3-dimensional imaging of an industrial catalyst, Mo-promoted colloidal Pt supported on carbon. The distribution of both the active Pt species and Mo promoter have been mapped over a single particle of catalyst using microfocus X-ray fluorescence computed tomography. X-ray absorption near edge spectroscopy (XANES) and extended X-ray absorption fine structure revealed a mixed local coordination environment, including the presence of both metallic Pt clusters and Pt chloride species, but also no direct interaction between the catalyst and Mo promoter. We also report on the benefits of scanning µ-XANES computed tomography for chemical imaging, allowing for 2- and 3-dimensional mapping of the local electronic and geometric environment, in this instance for both the Pt catalyst and Mo promoter throughout the catalyst particle.

Surface and interstitial Ti diffusion at the rutile TiO(2)(110) surface.

Diffusion of Ti through the TiO(2)(110) rutile surface plays a key role in the growth and reactivity of TiO(2). To understand the fundamental aspects of this important process, we present an analysis of the diffusion of Ti ad-species at the stoichiometric TiO(2)(110) surface using complementary computational methodologies of density functional theory corrected for on-site Coulomb interactions (DFT + U) and a charge equilibration (QEq) atomistic potential to identify minimum energy pathways. We find that diffusion of Ti from the surface to subsurface (and vice versa) follows an interstitialcy exchange mechanism, involving exchange of surface Ti with the 6-fold coordinated Ti below the bridging oxygen rows. Diffusion in the subsurface between layers also follows an interstitialcy mechanism. The diffusion of Ti is discussed in light of continued attempts to understand the re-oxidation of non-stoichiometric TiO(2)(110) surfaces.

Structure of adsorbed organometallic rhodium: model single atom catalysts.

We have determined the structure of a complex rhodium carbonyl chloride [Rh(CO)2Cl] molecule adsorbed on the TiO2(110) surface by the normal incidence x-ray standing wave technique. The data show that the technique is applicable to reducible oxide systems and that the dominant adsorbed species is undissociated with Rh binding atop bridging oxygen and to the Cl found close to the fivefold coordinated Ti ions in the surface. A minority geminal dicarbonyl species, where Rh-Cl bond scission has occurred, is found bridging the bridging oxygen ions forming a high-symmetry site.

Plasminogen expression in the neonatal and adult mouse brain.

Tissue-type plasminogen activator (tPA) has been implicated in a variety of types of neural plasticity, including cell migration, occlusion-induced visual system plasticity, and learning. In the periphery, plasminogen serves as tPA's primary substrate; however, studies attempting to identify plasminogen in the central nervous system have produced mixed results. We have performed a comprehensive, multitechnique study examining plasminogen expression in the neonatal and adult mouse brain. Reverse transcription polymerase chain reaction (RT-PCR) and in situ hybridization reveal plasminogen mRNA in the cortex, hippocampus and cerebellum of both neonatal and adult C57BL/6 mice. Immunocytochemistry reveals plasminogen protein expression in these same brain regions. Notably, plasminogen expression in the cerebellum occurs in the granule cell and the Purkinje cell layers. tPA activity in these same regions is involved in granule cell migration during development and motor learning in adulthood. Therefore, these findings demonstrate that plasminogen is present in the central nervous system and localized to areas where it could serve as a substrate for plasticity-related increases in tPA activity.

Early sensory and hormonal experience modulate age-related changes in NR2B mRNA within a forebrain region controlling avian vocal learning.

Male zebra finches are most apt to mimic songs heard between posthatch days (PHD) 35 and 65, and this vocal learning depends, in part, on the activation of N-methyl-D-aspartate receptors (NMDAR) within a discrete forebrain circuit that includes the lateral magnocellular nucleus of the anterior neostriatum (lMAN) and area X. Using in situ hybridization, we show that transcripts for both the constitutive NMDAR subunit NR1 and the modulatory subunit NR2B decrease abruptly in the lMAN between PHD20 and 40. This downregulation corresponds to the onset of song learning and a transition from slow to faster NMDAR currents in lMAN neurons. In area X, NR1 mRNA increases as NR2B mRNA decreases during song development. To understand how these changes in NMDAR mRNA might regulate song learning, we next investigated how manipulations that influence song development affect NMDAR mRNA expression. Early isolation from conspecific song (which delays closure of the sensitive period for song learning) selectively increases NR2B, but not NR1 mRNA, within lMAN at PHD60. In contrast, exposure to testosterone beginning at PHD20 (which impairs song development and hastens the developmental transition to faster NMDAR current kinetics within lMAN) accelerates the decline in NR2B mRNA in lMAN, again without affecting NR1 transcript levels. Neither manipulation significantly effects NR1 or NR2B mRNA levels in area X. Our data suggest that developmental changes in the expression of specific NMDAR subunits may regulate periods of neural and behavioral plasticity and that flexibility in the timing of these sensitive periods may be achieved through experience and/or hormone-dependent modulation of NMDAR gene expression.

Anticonvulsant prophylaxis and timing of seizures after aneurysmal subarachnoid hemorrhage.

OBJECTIVE: There is no evidence that seizure prophylaxis is indicated after aneurysmal subarachnoid hemorrhage (SAH). This study examines prophylactic antiepileptic drug (AED) prescription and the occurrence of seizures within a single university-affiliated institution. METHODS: The authors reviewed 95 SAH patient charts using standardized forms. Variables included prophylaxis duration, seizure incidence and timing, CT findings, AED adverse events, and 1-year patient follow-up. RESULTS: Prehospital seizures occurred in 17.9% (17/95) of patients; another 7.4% (7/95) had a questionable prehospital seizure. In-hospital seizures occurred in 4.1% (4/95) of patients, a mean of 14.5 +/- 13.7 days from ictus; three of these four patients were receiving an AED at the time of seizure. Inpatient AED were prescribed to 99% of the cohort for a median of 12 (range 1 to 68) days. Approximately 8% of the cohort had posthospital discharge seizures; this included the patients who had prehospital or in-hospital seizures, 50% of whom were receiving AED therapy at the time of the seizure. Adverse effects occurred in 4. 1%; none were serious. The thickness of cisternal clot was associated with having a seizure; no other clinical predictors were identified. Having a seizure at any time did not adversely affect outcome. CONCLUSIONS: In this SAH population, the majority of seizures happened before medical presentation. In-hospital seizures were rare and occurred more than 7 days postictus for patients receiving AED prophylaxis. The vast majority of putative clinical predictors did not help predict the occurrence of seizures; only the thickness of the cisternal clot was of value in predicting seizures. Patient selection for and the efficacy and timing of AED prophylaxis after SAH deserve prospective evaluation.

Neuronal migration is retarded in mice lacking the tissue plasminogen activator gene.

Neuronal migration is a critical phase of brain development, where defects can lead to severe ataxia, mental retardation, and seizures. In the developing cerebellum, granule neurons turn on the gene for tissue plasminogen activator (tPA) as they begin their migration into the cerebellar molecular layer. Granule neurons both secrete tPA, an extracellular serine protease that converts the proenzyme plasminogen into the active protease plasmin, and bind tPA to their cell surface. In the nervous system, tPA activity is correlated with neurite outgrowth, neuronal migration, learning, and excitotoxic death. Here we show that compared with their normal counterparts, mice lacking the tPA gene (tPA(-/-)) have greater than 2-fold more migrating granule neurons in the cerebellar molecular layer during the most active phase of granule cell migration. A real-time analysis of granule cell migration in cerebellar slices of tPA(-/-) mice shows that granule neurons are migrating 51% as fast as granule neurons in slices from wild-type mice. These findings establish a direct role for tPA in facilitating neuronal migration, and they raise the possibility that late arriving neurons may have altered synaptic interactions.

Developmental regulation of NMDA receptor 2B subunit mRNA and ifenprodil binding in the zebra finch anterior forebrain.

In passerine songbirds, song learning often is restricted to an early sensitive period and requires the participation of several discrete regions within the anterior forebrain. Activation of N-methyl-D-aspartate (NMDA) receptors is implicated in song learning and in one forebrain song region, the lateral magnocellular nucleus of the anterior neostriatum (IMAN), NMDA receptors decrease in density, their affinity for the antagonist MK-801 increases, and their currents decay more quickly as young male zebra finches lose the ability to imitate new song elements. These developmental changes in NMDA receptor pharmacology and physiology suggest that the subunit composition of NMDA receptors changes developmentally. Here, we have used in situ hybridization and [3H]ifenprodil receptor autoradiography to study the developmental regulation of the NMDA receptor 2B subunit (NR2B) within the anterior forebrain of male zebra finches. NR2B mRNA expression within the IMAN was twice as great in 30-day-old males (early in the sensitive period for song learning) as in adult males, and this developmental decrease in NR2B mRNA expression was mirrored by a decrease in high-affinity (NR2B-associated) [3H]ifenprodil binding within this song region. In another anterior forebrain song region, Area X, NR2B mRNA also declined significantly after 30 days posthatch, but this decline was not accompanied by a significant decrease in [3H]ifenprodil binding. The results are consistent with the hypothesis that developmental changes in NMDA receptor function mediated by regulation of subunit composition contribute to the sensitive period for vocal learning in birds.

Blockade of NMDA receptors in the anterior forebrain impairs sensory acquisition in the zebra finch (Poephila guttata).

Juvenile zebra finches (Poephila guttata) learn song in two stages: during sensory acquisition, they memorize the song of an adult tutor, and during sensorimotor learning, they alter their vocalizations to match the stored song model. Like many other forms of neural plasticity and memory formation, vocal learning in zebra finches is impaired by pharmacological blockade of NMDA receptors, but the relevant NMDA receptors have not yet been localized. During song development, one neural region that has been implicated specifically in song learning, the IMAN, exhibits an increased density of NMDA receptors as well as decreased binding affinity for the NMDA antagonist MK-801. To test the hypothesis that sensory acquisition requires activation of NMDA receptors in or near the IMAN we infused the NMDA receptor antagonist amino-5-phosphonopentanoic acid (AP5; 2.5 micrograms 0.1 microliter) directly into the anterior forebrain. Birds receiving AP5 infusions prior to each of 10 tutoring sessions copied significantly less of their tutor's song than did sham-operated birds, saline-infused birds, birds that received AP5 infusions on nontutoring days, or birds that received AP5 infusions into the cerebellum. Furthermore, infusions of AP5 in the anterior forebrain did not impair young birds' ability to discriminate zebra finch from canary song. These findings are consistent with the hypothesis that NMDA receptor activation in the anterior forebrain is necessary for the memorization of song material during avian vocal learning. This is also the first report that song-related regions of the anterior forebrain contribute to sensory acquisition specifically.

Behavioral effects of 3 alpha-androstanediol. II: Hypothalamic and preoptic area actions via a GABAergic mechanism.

We investigated whether 5 alpha-androstane-3 alpha, 17 beta-diol (3 alpha-androstanediol; 3 alpha-Diol), a neurosteroid whose effects are primarily inhibitory to sexual behavior, may act through interactions with gamma-aminobutyric acid (GABA) receptor complexes (GBRs) in the medial basal hypothalamus (MBH) and the preoptic area (POA). In Experiment (Exp.) 1, ovariectomized (ovx) rats were implanted with bilateral guide cannulae aimed above the MBH and were later treated with 17 beta-estradiol (E2, 2 injections of 1 microgram/0.2 ml in 10% ethanol) and either 3 alpha-Diol (3.0 mg/kg, s.c.) or vehicle. Progesterone (0.5 mg, s.c.) was given 24 h after the first E2 injection and a pre-test for lordosis responsiveness was carried out 4 h later. The GABAA agonist, muscimol (50 ng), then was infused into the MBH and rats were tested 10, 30 and 60 min later. Muscimol infusion facilitated lordosis behavior in vehicle-treated controls, but 3 alpha-Diol-treated animals failed to show this facilitation. To ascertain whether 3 alpha-Diol would also prevent muscimol's action in the POA, a site in which muscimol inhibits, rather than facilitates, sexual receptivity, ovx animals in Exp.2 were implanted with bilateral guide cannulae aimed above the POA and were treated with E2, 3 alpha-Diol, and P and infused and tested as in Exp. 1. Muscimol and 3 alpha-Diol each significantly inhibited receptivity; when they were combined, the inhibition was more pronounced. In Exp. 3, POA infusions of the GABAA antagonist, bicuculline, counteracted muscimol's and 3 alpha-Diol's inhibition of sexual behavior. In Exp. 4, in vitro treatment of POA and MBH membrane fractions with 3 alpha-Diol (30 microM) enhanced maximal [3H]muscimol binding without altering the affinity of the binding sites for the agonist. These data suggest that 3 alpha-Diol inhibits E2 and progestin-induced lordosis behavior via actions at the GBR in both the MBH and POA.

Estrogen receptor immunoreactive neurons in the fetal ferret forebrain.

The development of estrogen receptors was studied in the preoptic area/anterior hypothalamus (POA/AH) of fetal male and female ferrets. In males this region includes a nucleus (MN-POA/AH), delineated by Nissl stains, which is not discernible in females. The results reveal the distribution of estrogen receptor containing cells during the period when estrogen is known to induce the differentiation of the male ferret's MN-POA/AH. Brains were taken from ferret kits on days 30, 34, 37 and 40 of a 41-42 day gestation, and were processed utilizing the H222 monoclonal antibody to reveal estrogen receptors. At E30 there were numerous H222 immunoreactive (ir) cells in central regions of the POA/AH. From E30 to E40 there was a striking increase in the number of H222ir cells in the POA/AH. A broad sweep of H222ir cells extended from the ventral POA dorsally and laterally into the caudal POA and AH of both males and females. H222ir cells were not restricted to the region of the MN-POA/AH at any fetal age. H222 immunoreaction product at E30 was restricted to nuclear compartments. By E40, H222ir processes extended from some cells with H222ir nuclei in the medial and lateral POA/AH in both males and females. At the older fetal ages immunopositive cell numbers increased in lateral positions. At E34 and E37 (but not E30) selective ventricular zones, and regions between the hypothalamus and amygdala contained H222ir cells, suggesting the presence of estrogen receptors in cells during migration. Although the amygdala contained a few H222ir cells as early as E34, the cortex lacked H222ir cells even as late as E40. The appearance of H222ir cells in positions suggestive of migration is consistent with the hypothesis that estrogen receptors play some role in determining cell positions in certain regions of the developing nervous system.

Neonatal testosterone masculinizes sexual behavior without affecting the morphology of the dorsal preoptic/anterior hypothalamic area of female ferrets.

We examined whether testosterone (T) administered to female ferrets neonatally--a treatment known to enhance masculine coital capacity--induces formation of the sexually dimorphic male nucleus in the dorsal preoptic/anterior hypothalamic area (MN-POA/AH), and/or sensitizes dorsal POA/AH neurons to the stimulatory effect of later androgen treatment on somal dimensions. In males, the MN-POA/AH was present in all subjects, and exposure to androgen following castration at postnatal day 56 (P56) increased both MN-POA/AH volume as well as mean somal areas of MN-POA/AH neurons relative to oil-treated controls. Females given androgen from P5 to P20 and for one month beginning after ovariectomy on P56 failed to develop the MN-POA/AH, but displayed high levels of masculine sexual behavior. Somal areas of dorsal POA/AH neurons in females that received either T or a control neonatally did not increase following androgen treatment at P56. Thus, the correlation that exists between somal enlargement of dorsal POA/AH neurons and masculine sexual behavior in androgen-treated males is not found in behaviorally masculinized females. Masculine coital ability does not appear related to aspects of dorsal POA/AH morphology, supporting data from a previous study in which lesions of the MN-POA/AH caused negligible deficits in masculine sexual behavior of adult male ferrets.

Ontogeny of the sexually dimorphic male nucleus in the preoptic/anterior hypothalamus of ferrets and its manipulation by gonadal steroids.

A sexually dimorphic nucleus exists in the dorsal region of the ferret preoptic/anterior hypothalamic area (POA/AH), and is called the male nucleus of the POA/AH (MN-POA/AH) because it is found only in males. Development of the MN-POA/AH was studied in male ferrets, and for comparison a sexually nondimorphic ventral POA/AH nucleus was studied in both sexes. The MN-POA/AH was conspicuous in males as early as embryonic day 37 (E37) of a 41-day gestation, and its volume increased until postnatal day 56 (P56). No nucleus was present in the dorsal POA/AH of females at any age. The densities and average somal areas of cells in the dorsal POA/AH were similar in males and females at E33, before the MN-POA/AH could be visualized. However, at E37 and E41 dorsal cells were greater in density and/or somal area in males than in females, accounting for the appearance of a nucleus in males at these ages. To insure that the dorsal POA/AH nucleus seen in males at E37 and E41 was the presumptive MN-POA/AH present in adult males, pregnant ferrets were given progesterone and either implanted subcutaneously (s.c.) with testosterone (T) or ovariectomized and implanted s.c. with the aromatase inhibitor, 1,4,6-androstatriene-3,17-dione (ATD), on day 30 of gestation. As predicted from previous studies in which subjects were sacrificed in adulthood, formation of a dorsal POA/AH nucleus was promoted in female ferrets by T, and blocked in males by maternal ovariectomy and ATD treatment for animals sacrificed at E41. Much evidence suggests that behavioral sexual differentiation is accomplished in the male ferret between age E28 and P20. The MN-POA/AH is present and potentially functional in males during a considerable portion of this perinatal period.